C1ql3/BAI1 Signal Promotes Synapse Maturation Of The Neuromuscular Junction

Backgroud and objective: The neuromuscular junction(NMJ)is the synaptic structure between motor neurons and skeletal muscles.Motor neuron axon terminal releases neurotransmitter acetylcholine(ACh)to bind to the acetylcholine receptor(AChR)on the postsynaptic membrane.The binding triggers the end-plate potential,leading to the generation of muscle action potential and contraction.Only through the NMJ,the nervous system controls the body movement.Defects in the NMJ cause numerous neural diseases,damage our health,and even threaten lives.The NMJ shares the similar structural and functional characteristics with the central nervous system synapse,meanwhile it is relatively easier to manipulate,so the NMJ works as a classic model to study the mechanisms of synapse formation and development,and helps to understand the central nervous system synapse formation,development,and maturation.Although the core regulatory signal of the NMJ formation had been identified,we still knew little about the mechanisms of the NMJ maturation.Synapse elimination and AChR clusters dispersion are hallmark steps of the NMJ maturation.Recent reports found that complement and related proteins play essential roles in the synapse elimination of the central nervous system neurons.In a way similar to their functions in the immune system,complement proteins,such as C1 q,could label the synapses that need to be removed in the developing central nervous system.Microglia then recognized the label using complement receptors and eliminate these synapses.Astrocyte could secret signals to induce the expression of complement.C1ql(C1q like),another member of the C1 q family,also had been reported involved in the maturation of the central nervous system synapse.However,the function and mechanism of complement related proteins in the NMJ maturation remained unclear.Therefore,this study aimed to clarify the role of C1 q protein in NMJ maturation.Methods: We first used RT-PCR to measure the expressions of the C1ql1-4 and receptors BAI1-3(brain-specific angiogenesis inhibitor 1-3)in the neuromuscular tissues at different developmental stages and in cultured Schwann cells and muscle cells.To investigate the role of C1 ql protein in the depolymerization of AChR,We used agrin-induced AChR clustering in the muscle cells as a model to explore the function of C1 ql in AChR clusters dispersion.To examine whether this dispersing effect of C1 ql was mediated by its receptor BAI,we generated BAI1-3 genes mutant muscle cell lines by the CRISPR-cas9 technique.We further confirmed the interaction between BAI1 and downstream protein ELMO1(Engulfment and cell motility 1)using immunoprecipitation assay.In addition,a blocking peptide was used to block the interaction between BAI1 and ELMO1 to explore whether this interaction was essential in the depolymerization of AChR clusters promoted by C1ql3.In the mice experiment,we overexpressed C1ql3 or BAI1 by electrotransfection in the anterior tibial muscle to enhance or inhibit C1ql3/BAI1 signal and observed the morphological changes of NMJ in synaptic maturation phase by confocal microscopy.Results:1.C1ql1-4 were expressed in the spinal cord of mice at different developmental stages and Schwann cells cultured in vitro,and its receptor BAI1-3 were also expressed in the neuromuscular tissues of mice at different developmental stages and muscle cell lines cultured in vitro.2.In vitro cultured myotubes,the aggregation of AChR stimulated by agrin would depolymerize after the removal of agrin.C1 ql protein could promote the depolymerization of AChR aggregation induced by agrin in myocytes.The depolymerization effect of C1ql3 was dose-dependent,and the depolymerization effect increased with the increase of C1ql3 concentration.3.BAI3 knockout could affect the fusion of myoblasts and BAI1,and BAI2 knockout did not affect the aggregation of AChR induced by agrin.The mutation of BAI1 could block the depolymerization of AChR promoted by C1ql3,indicating that the depolymerization of AChR promoted by C1ql3 was mediated by the receptor BAI1 protein.4.Blocking the interaction between BAI1 and ELMO1 by polypeptide could block the binding of BAI1 and ELMO1,thus inhibiting the effect of C1ql3 on promoting AChR depolymerization,indicating that the intracellular protein ELMO1 was involved in promoting AChR depolymerization by C1ql3 / BAI1.5.The density and area of AChR in muscle fibers of C1ql3 overexpression mice were significantly higher than those of control mice,and the number of multiinnervation was significantly lower than that of control mice of the same age,suggesting the acceleration of synaptic maturation.The density and area of AChR were less than that of control,and the number of muscle fiber multiinnervation was significantly higher than that of C1ql3 overexpression mice and control mice,indicating delayed synaptic maturation.Conclusion: C1ql3 could recognize and bind to muscle cell membrane protein BAI1 to activate downstream signal,and promote AChR depolymerization in muscle cells,thus playing an important role in the maturation of the NMJ.